In order to realize the potential of gene therapy, the major obstacle of gene silencing must be overcome. In gene therapy, after good copies of a defective gene are delivered to the appropriate cells, the genes encoded in the DNA quickly become silenced. This silencing is due to the reconfiguration of chromatin on the introduced DNA. Initially, nucleosomes form on the DNA in a euchromatic conformation that allows appropriate gene expression, but over time, nucleosome positions shift to a heterochromatic conformation that silences the genes. Our group and others have recently shown that the underlying DNA sequence can have a large impact on nucleosome positions, chromatin structure and gene expression. We propose to address the problem of gene silencing by developing strategies for engineering nucleosome positioning information into transgene DNA. The novel unc-54 enhancer/myo-2 promoter combination in C. elegans faithfully recapitulates a generational gene-silencing phenomenon. The enhancer-induced transgene expression is strong in the first generation of transgenic animals, but is then silenced in subsequent generations. We will use this convenient promoter/enhancer combination to test the effects of engineered nucleosome positioning elements on generational gene silencing. The redesigned chromatin architectures will be evaluated by both transgene expression and direct nucleosome mapping by a novel pull-out assay. Finally, we will use a forward genetic screen to find the factors that affect somatic gene silencing. Hypothesis: Transgene silencing due to chromatin changes in nucleosome positions can be overcome by engineering vectors with nucleosome positioning and repelling sequences to position and anchor nucleosomes in conformations that are amenable to gene expression. To test this hypothesis we propose three specific aims: Aim 1: Engineer and test nucleosome positioning transgenes for long-term gene expression. By modifying the unc-54 enhancer/myo-2 promoter construct through inserting nucleosome positioning and repelling elements, we will engineer transgenes that maintain long-term, multigenerational expression in C. elegans. Aim 2: Determine nucleosome positioning on extrachromosomal transgene arrays to validate modified chromatin architecture. We will biochemically validate our genetic results from Aim 1 by developing a cell-specific transgene pull-out system. We will evaluate nucleosome positioning and chromatin architecture using MNase-Seq thus confirming the reconformation of nucleosomes on our engineered constructs. Aim 3: Identify somatic factors that cause gene silencing. We will identify native genes in C. elegans that modulate the transgene silencing phenomenon by screening for mutants in which transgene silencing is attenuated, and subsequent identification of associated genes.